HIP1R acts as a tumor suppressor in gastric cancer by promoting cancer cell apoptosis and inhibiting migration and invasion through modulating Akt

Abstract Background Huntingtin‐interacting protein 1‐related (HIP1R) is a multi‐domain gene that exerts many cellular functions including altering T cell–mediated cytotoxicity and controlling intracellular trafficking. However, its clinical significance and function in gastric cancer (GC) have not been described. Methods The expression levels of HIP1R were tested by the transcriptional and translational expression analysis and immunohistochemistry (IHC) in matched adjacent non‐tumorous vs tumor tissue specimens. The biological function of HIP1R on apoptosis, migration, and proliferation was evaluated by flow cytometry, Transwell, Cell Counting Kit‐8 (CCK‐8) assays, colony formation assays, and EdU labeling assays, respectively. Results We found downregulated HIP1R in GC compared with adjacent non‐tumorous tissue, and HIP1R expression associated with N classification. We further found that the expression of HIP1R could induce apoptosis and inhibit proliferation, migration, invasion of GC cells, possibly through modulating Akt. Conclusions Our data indicate that HIP1R may act as a potential diagnostic biomarker and a tumor suppressor gene in GC, potentially representing a novel therapeutic target for future GC treatment.

Huntingtin-interacting protein 1-related (HIP1R) 4,5 belongs to an evolutionarily conserved family 6 along with yeast Sla2p 7,8 and mammalian HIP1 proteins. 9 HIP1 and HIP1R are reported to be components of the clathrin-mediated endocytosis pathway and have known functions in endocytosis and actin cytoskeleton regulation. [10][11][12][13][14][15] HIP1R is the only known mammalian relative of HIP1, and previous reports indicate that HIP1 and HIP1R expression alterations may contribute to cell growth and survival. 16 HIP1 has been reported to affect tumorigenesis in many tumor types; [17][18][19][20][21] however, HIP1R has rarely been reported on in tumors. Previous studies suggest that HIP1R may be a key factor in maintaining chromosome integrity.
In HIP1R-deficient cells, chromosomes mismatched and eventually produced multinucleated cells, indicating that HIP1R is essential for tumorigenesis or other human diseases. 22 Additionally, recent studies found that HIP1R plays a significant role in tumor immunotherapy by modulating T cell-mediated cytotoxicity via lysosomal degradation of PD-L1. 23 Low expression of HIP1R is associated with worse prognosis in diffuse large B-cell lymphoma (DLBCL) patients with the treatment of rituximab-CHOP regimens. 24,25 Ectopic HIP1R expression has also been demonstrated to promote migration and invasion of less-invasive prostate cancer cells. 26 Although HIP1R associates with human cancer biology, its precise role in GC progression remains unclear. Therefore, our study was designed to explore the clinicopathological associations, biological functions, and possible regulatory mechanisms of HIP1R in GC.

| Patients and samples
All patient specimens were gathered with signed informed consent from the patients, and ethics approval for the study was gained  Table 1.

| Cell cultivation and transfection
The cells used in this study included AGS, HGC27, SGC7901, MGC803, and one normal gastric epithelium cell line (GES1). All cells were obtained from the cell line library of the Chinese Academy of Sciences. The cell culture conditions were as follows: the medium was RPMI 1640 medium (Invitrogen) with 10% fetal bovine serum, the temperature in the wet incubator was 37°C, and it contained 5% carbon dioxide (Thermo).
For HIP1R overexpression, the CMV-MCS-IRES-EGFP-SV40-Neomycin plasmid was obtained from Genechem. Empty vector was used as a negative control. The small interfering RNA (siRNA) against HIP1R and control siRNA were obtained from Syngen Tech.
GC cells were transfected with plasmid or with HIP1R siRNA using lipofectamine 3000 Reagent (Invitrogen) following the instructions on the manual. Cells were collected 48 hours after transfection.
Quantitative real-time PCR (qRT-PCR) or Western blotting was used to monitor transfection efficiency.

| Western blotting
Total proteins from transfected GC cells were extracted via the Total Protein Extraction Kit (KeyGEN). The BCA protein assay was performed for protein quantification from the total extracted cell protein lysates, then the same-volume of protein samples were added into the SDS-PAGE and transferred onto PVDF membranes (Millipore).
The proteins on the membrane were then blocked with 5% skim milk for 2 hours and incubated overnight at 4°C with anti-HIP1R

| Tissue microarrays (TMAs) and IHC
The representative areas of each formalin-fixed paraffin-embedded tissue cylinder (typical diameter: 2 mm) were punched from different primary GC blocks (cores of intratumoral or peritumoral tissues from "donor" blocks) and collected into one empty "recipient" wax block like a microarray (4 cm × 2 cm) as previously described. 27 The median age at surgery for this cohort was 60 y. b The median size of the primary tumor for this cohort was 5 cm.

| Colony formation assays
Cells were plated at 6-well plates (1000 cells/well) and incubated for 1 week. The cells were fixed with methanol for 10 minutes and then stained with 1% crystal violet. Colonies were manually counted for more than 50 cells using ImageJ. The experiment was carried out at least three times.

| EdU labeling assays
Cell proliferation was determined using the BeyoClick™ EdU Kit (Beyotime Biotechnology) following the manufacturer's instructions.
Briefly, cells were plated at 12-well plates (1 × 10 5 cells/well) and incubated with equal volumes of the EdU work solution for 2 hours at 37°C. Then, cells were fixed with 4% paraformaldehyde for 15 minutes. The stained cells were examined with Leica LMD6500 fluorescence microscope and randomly photographed with 10 fields.

| Transwell assays
Cell migration and invasion abilities were measured using HGC27 and  and stained with hematoxylin and eosin. Ten fields were randomly imaged from the underside of the membrane using a Leica DM4000B microscope and the number of cells was counted with ImageJ.

| Flow cytometry
Apoptosis was measured using the manufacturer's method (KeyGEN).
Briefly, cells were collected 48 hours after transfection then washed with PBS. Annexin V APC and propidium iodide (PI) staining were used to assess the apoptosis rate of cancer cells within 30 minutes after staining. Each sample was then analyzed by BD FACSCalibur flow cytometry system (BD) using Cell-Quest software.

| HIP1R is downregulated in GC
Since little was published about HIP1R in GC, we decided to analyze open datasets from the Oncomine database and found downregulated HIP1R mRNA expression in human GC samples compared to normal gastric samples ( Figure 1A).  Figure 2A-D).

| Diagnostic value of HIP1R for GC patients
To compare the diagnostic capacity of HIP1R between controls and GC, we performed a corresponding receiver operating characteristic (ROC) curve. As shown in Figure 2E

| HIP1R promotes GC cell apoptosis
To explore the function of HIP1R in GC, we performed qRT-PCR to

| HIP1R inhibits GC cell proliferation
To investigate the proliferation function of HIP1R in GC, we performed the Cell counting kit-8 (CCK-8) assays, colony formation assays and EdU labeling assays. The results were consistent and all showed that HIP1R overexpression inhibited proliferation rate of HGC27 cells, while HIP1R siRNA caused increased proliferation rate of MGC803 cells ( Figures 4E,F and 5A).

| HIP1R inhibits GC cell motility
To examine the effect of HIP1R on GC cell migration and invasion ability, we performed Transwell assay analysis. We found that HIP1R overexpression restricted GC cell migration and invasion ability compared with cells transfected with negative control. Transwell assays further showed an increased invasion ability in GC cells transfected with HIP1R siRNA compared with control siRNA group ( Figure 5B).

| The PI3K/Akt signaling pathway may be regulated by HIP1R
To investigate pathways participating in HIP1R-mediated GC progression, GSEA was performed using published GC data from the TCGA GC database (n = 375). We found that the PI3K/Akt signal transduction pathway most correlated with HIP1R expression ( Figure 6A). To verify this analysis result, we examined changes in Akt, phospho-Akt (p-Akt), mTOR, phospho-mTOR (p-mTOR), Cleaved Caspase-9 and Bak by Western blotting. We found that compared with the empty vector group, the expression levels of p-Akt, p-mTOR were suppressed in the HIP1R plasmid group, but the expression levels of Cleaved Caspase-9, Bak were increased.
Opposing results were observed in the HIP1R siRNA group compared with the control siRNA group ( Figure 6B). To investigate whether HIP1R suppresses epithelial-mesenchymal transition (EMT) via the AKT pathway, we also examined changes of E-Cadherin and Vimentin. We found that overexpression of HIP1R enhanced the expression of E-cadherin and reduced the expression of Vimentin, and vice versa ( Figure 6C). To further ascertain the relationship between HIP1R and p-Akt, we performed the IHC in TMA in 73 paired samples. We found that the expression of HIP1R inversed correlated with the expression of p-Akt ( Figure 6D,E). expression has properties to inhibit cancer cell migration and invasion.

| CON CLUS ION
In conclusion, our studies indicate that HIP1R is downregulated in human GC in association with N stage. We also demonstrated the diagnostic efficiency of HIP1R in GC. Furthermore, the expression of HIP1R could promote GC cell apoptosis and inhibit proliferation, migration and invasion, possibly through modulating Akt. We believe that HIP1R downregulation may identify high-risk patients and serve as a new therapeutic target for GC.

ACK N OWLED G M ENTS
The authors would like to acknowledge the Key Laboratory of